Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Monophasic expression of FliC by Salmonella 4,[5],12:i:- DT193 does not alter its pathogenicity during infection of porcine intestinal epithelial cells.

Non-typhoidal serotypes of Salmonella enterica remain important food-borne pathogens worldwide and the frequent emergence of epidemic strains in food-producing animals is a risk to public health. In recent years, Salmonella 4,[5],12:i:- isolates, expressing only phase 1 (FliC) of the two flagellar antigens, have emerged and increased in prevalence worldwide. In Europe, the majority of 4,[5],12:i:- isolates belong to phage types DT193 and DT120 of Salmonella Typhimurium and pigs have been identified as the reservoir species. In this study we investigated the ability of pig-derived monophasic (4,[5],12:i:-) and biphasic DT193 isolates to invade a porcine intestinal epithelial cell line (IPEC-1) and activate TLR-5, IL-8 and caspases. We found that the 4,[5],12:i:- isolates exhibited comparable adhesion and invasion to that of the virulent S. Typhimurium isolate 4/74, suggesting that these strains could be capable of colonizing the small intestine of pigs in vivo. Infection with 4,[5],12:i:- and biphasic DT193 isolates resulted in approximately the same level of TLR-5 (a flagellin receptor) and IL-8 (a proinflammatory chemokine) mRNA upregulation. The monophasic variants also elicited similar levels of caspase activation and cytotoxicity to the phase-variable DT193 isolates. These findings suggest that failure of 4,[5],12:i:- DT193 isolates to express a second phase of flagellar antigen (FljB) is unlikely to hamper their pathogenicity during colonization of the porcine intestinal tract.

Chryseobacterium zeae sp. nov., Chryseobacterium arachidis sp. nov., and Chryseobacterium geocarposphaerae sp. nov. isolated from the rhizosphere environment.

Four yellow pigmented strains (91A-561(T), 91A-576, 91A-593(T), and JM-1085(T)) isolated from plant materials, showed 97.2-98.7 % 16S rRNA gene sequence similarities among each other and were studied in a polyphasic approach for their taxonomic allocation. Cells of all four isolates were rod-shaped and stained Gram-negative. Comparative 16S rRNA gene sequence analysis showed that the four bacteria had highest sequence similarities to Chryseobacterium formosense (97.2-98.7 %), Chryseobacterium gwangjuense (97.1-97.8 %), and Chryseobacterium defluvii (94.6-98.0 %). Sequence similarities to all other Chryseobacterium species were below 97.5 %. Fatty acid analysis of the four strains showed Chryseobacterium typical profiles consisting of major fatty acids C15:0 iso, C15:0 iso 2-OH/C16:1 ω7c, C17:1 iso ω9c, and C17:0 iso 3-OH, but showed also slight differences. DNA-DNA hybridizations with type strains of C. gwangjuense, C. formosense, and C. defluvii resulted in values below 70 %. Isolates 91A-561(T) and 91A-576 showed DNA-DNA hybridization values >80 % indicating that they belonged to the same species; but nucleic acid fingerprinting showed that the two isolates represent two different strains. DNA-DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed, that both strains 91A-561(T) and 91A-576 represent a novel species, for which the name Chryseobacterium geocarposphaerae sp. nov. (type strain 91A-561(T)=LMG 27811(T)=CCM 8488(T)) is proposed. Strains 91A-593(T) and JM-1085(T) represent two additional new species for which we propose the names Chyrseobacterium zeae sp. nov. (type strain JM-1085(T)=LMG 27809(T), =CCM 8491(T)) and Chryseobacterium arachidis sp. nov. (type strain 91A-593(T)=LMG 27813(T), =CCM 8489(T)), respectively.

O antigen is the receptor of Vibrio cholerae serogroup O1 El Tor typing phage VP4.

Bacteriophage VP4 is a lytic phage of the Vibrio cholerae serogroup O1, and it is used in phage subtyping of V. cholerae biotype El Tor. Studies of phage infection mechanisms will promote the understanding of the basis of phage subtyping as well as the genetic differences between sensitive and resistant strains. In this study, we investigated the receptor that phage VP4 uses to bind to El Tor strains of V. cholerae and found that it infects strains through adsorbing the O antigen of V. cholerae O1. In some natural isolates that are resistant to VP4 infection, mutations were identified in the wb* cluster (O-antigen gene cluster), which is responsible for the biosynthesis of O antigen. Mutations in the manB, wbeE, and wbeU genes caused failure of adsorption of VP4 to these strains, whereas the observed amino acid residue mutations within wbeW and manC have no effect on VP4 infection. Additionally, although mutations in two resistant strains were found only in manB and wbeW, complementing both genes did not restore sensitivity to VP4 infection, suggesting that other resistance mechanisms may exist. Therefore, the mechanism of VP4 infection may provide a basis for subtyping the phage. Elaborate mutations of the O antigen may imbue V. cholerae strains with resistance to phage infection.

Polyphasic characterization of Salmonella Enteritidis isolates on persistently contaminated layer farms during the implementation of a national control program with obligatory vaccination: a longitudinal study.

Since 2007, a national Salmonella control program including obligatory vaccination has been ongoing in Belgium. In this context, the aim of the present study was to investigate the diversity of Salmonella enterica serovar Enteritidis isolates on 5 persistently contaminated Belgian layer farms and to examine the potential sources and transmission routes of Salmonella Enteritidis contamination on the farms during successive laying rounds. A collection of 346 Salmonella isolates originating from the sampled farms were characterized using a combination of multilocus variable number of tandem repeat analysis (MLVA) and phage typing (PT). On each farm, one or 2 dominant MLVA-PT types were found during successive laying cycles. The dominant MLVA type was different for each of the individual farms, but some farms shared the same dominant phage type. Isolates recovered from hens' feces and ceca, egg contents, eggshells, vermin (mice, rats, red mites, and flies), and pets (dog and cat feces) had the same MLVA-PT type also found in the inside henhouse environment of the respective layer farm. Persistent types were identified in the layer farm inside environment (henhouse and egg collecting area). Furthermore, this study demonstrated cross-contamination of Salmonella between henhouses and between the henhouse and the egg collecting area. Additional isolates with a different MLVA-PT type were also recovered, mainly from the egg collecting area. A potential risk for cross-contamination of Salmonella between the individual layer farms and their egg trader was identified.

Prevalence of R-type ACSSuT in strains of Salmonella serovar Typhimurium DT193 isolated from human infections in Brazil / Prevalencia de resistencia de tipo ACSSuT en cepas de Salmonella serovariedad Typhimurium DT193 aisladas a partir de infecciones humanas en el Brasil

OBJECTIVE: To determine the prevalence of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT) in Salmonella serovar Typhimurium definitive [phage] type (DT) 193 strains isolated from human sources over the last four decades. METHODS: From 2008 to 2010, 553 DT193 isolates out of 810 human-origin Salmonella ser. Typhimurium phage-typed strains isolated from the 1970s through 2008 were selected and tested for ACSSuT resistance: 91 strains isolated during the 1970s, 65 from the 1980s, 70 from the 1990s, and 327 from 2000-2008. Resistance profiles were determined using the disk diffusion method. RESULTS: †An antimicrobial susceptibility assay indicated 20.9 percent, or 116, of all isolates tested were ACSSuT-resistant, 52.0 percent (287) were resistant to one or more drugs in the ACSSuT profile, and 27.1 percent (150) were nonresistant (susceptible to antimicrobials). Based on the assay, overall antimicrobial resistance was extremely high in the 1970s (affecting 99.0 percent of isolates from that period) and remained high during the 1980s, when 95.4 percent of isolates had some type of antimicrobial resistance and incidence of Salmonella ser. Typhimurium DT193 R-type ACSSuT increased to 73.8 percent. R-type ACSSuT dropped to 27.1 percent (19 isolates) during the 1990s, and to 5.2 percent (17) during 2000-2008, despite a substantial increase in the number of isolates tested (397 versus 204, 111, and 98, respectively, for the previous three decades). CONCLUSIONS: †Although prevalence of Salmonella ser. Typhimurium DT193 R-type ACSSuT in Brazil has decreased since the 1970s, ACSSuT resistance markers continue to circulate. Therefore, continuous surveillance should be conducted to evaluate the occurrence of Salmonella ser. Typhimurium DT193 and its antimicrobial resistance.

OBJETIVO: Determinar la prevalencia de resistencia a la ampicilina, el cloranfenicol, la estreptomicina, las sulfonamidas y las tetraciclinas (ACSSuT) en cepas de Salmonella serovariedad Typhimurium fagotipo definitivo (DT) 193 aisladas de fuentes de origen humano durante las cuatro últimas décadas. MÉTODOS: Entre el 2008 y el 2010 se seleccionaron 553 aislados de DT193 entre 810 cepas de Salmonella serovariedad Typhimurium fagotipificadas aisladas desde la década de 1970 hasta el 2008, y en ellos se analizó la resistencia a ACSSuT: se estudiaron 91 cepas aisladas durante la década de 1970, 65 aisladas durante la década de 1980, 70 aisladas durante la década de 1990, y 327 aisladas entre el 2000 y el 2008, respectivamente. Los perfiles de resistencia a los antibióticos se determinaron mediante el método de difusión en disco. RESULTADOS: El antibiograma indicó que 20,9 por ciento (116) de todos los aislados que se analizaron fueron resistentes a ACSSuT, 52,0 por ciento (287) fueron resistentes a uno o más antibióticos del grupo ACSSuT y 27,1 por ciento (150) no fueron resistentes (es decir, fueron sensibles a dichos antibióticos). Según el análisis, la resistencia general a los antibióticos fue muy alta en la década de 1970 (y comprendió a 99,0 por ciento de los aislados de ese período) y continuó siendo alta durante la década de 1980, cuando 95,4 por ciento de los aislados presentó algún tipo de resistencia a los antibióticos y la incidencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT aumentó hasta 73,8 por ciento. La resistencia de tipo ACSSuT descendió a 27,1 por ciento (31 aislados) durante la década de 1990, y a 5,2 por ciento (17 aislados) entre el 2000 y el 2008, a pesar del aumento importante en el número de aislados que se evaluaron (397 frente a 204, 111 y 98 en las tres décadas anteriores, respectivamente). CONCLUSIONES: Aunque la prevalencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT en el Brasil ha disminuido desde la década de 1970, los marcadores de resistencia a ACSSuT continúan en circulación. Por consiguiente, debe llevarse a cabo una vigilancia permanente para evaluar la aparición de infecciones por Salmonella serovariedad Typhimurium DT193 y su resistencia a los antibióticos.

Characterisation of methicillin resistant S. aureus strains and risk factors for acquisition in a teaching hospital in northeast India.

PURPOSE: A point prevalence study was carried out in a teaching hospital in Assam to characterise S. aureus strains, establish the rate of colonisation of methicillin resistant S. aureus (MRSA) and associated risk factors for its acquisition. MATERIALS AND METHODS: Antibiogram-Resistogram profile was done by BSAC standardized disc sensitivity method; Phage and RFLP typing were carried out by the PHLS, London. RESULTS: Single MRSA strain resistant to multiple classes of anti-staphylococcal antibiotics dominated the hospital. The MRSA colonisation rate was found to be 34% (n=29) and 18% (n=80) in orthopaedics and surgery, respectively and only approximately 1% (n=73) in the medical units. Exposure to ciprofloxacin and surgery were risk factors but duration of hospital stay was not. In contrast, methicillin sensitive S. aureus (MSSA) strains were usually distinct strains and sensitive to most of the anti-staphylococcal antibiotics including 18% to penicillin. CONCLUSIONS: The MRSA strain prevalent in the hospital phenotypically resembles the predominant Asian strain viz., Brazilian/Hungarian strains (CC8-MRSA-III). Duration was not a risk factor, which suggests that in absence of exposure to specific antimicrobials, even in a hospital with no or little infection control intervention, a vast majority remain free from MRSA. This underlines the importance of rational prescribing empirical antibiotics.

Linezolid-resistant ST36 methicillin-resistant Staphylococcus aureus associated with prolonged linezolid treatment in two paediatric cystic fibrosis patients.

OBJECTIVES: To describe the emergence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) of sequence type (ST)36 lineage in two paediatric patients with cystic fibrosis, after long-term low-dose linezolid treatment. METHODS: Two paediatric males with cystic fibrosis had sputum samples quantitatively cultured during hospitalization. After the isolation of MRSA from both patients, oral treatment with 300 mg linezolid twice daily was initiated for periods of 1-2 months separated by up to 6 months. Isolates cultured 9 months after the start of treatment were tested for resistance to linezolid by agar dilution (BSAC). Resistant isolates were examined for 23S rDNA mutations, and typed by phage and macrorestriction with SmaI. Isolates from follow-up sputum samples were obtained until 44-51 months after treatment with linezolid. RESULTS: Colonization with MRSA was at a density of approximately 10(6) cfu/mL sputum for both subjects. Initial isolates were susceptible to linezolid, but, 9 months later, isolates from both patients were resistant (MICs > 16 mg/L). Both isolates were epidemic MRSA-16 variant A1 (ST36-MRSA-II), which is widespread in UK hospitals. Both isolates were heterozygous for a G2576T mutation in their 23S rDNA genes, but one was resistant to fusidic acid and tetracycline. In follow-up sampling, the younger patient yielded linezolid-resistant EMRSA-16 for a further 42 months, whilst the other lost the linezolid-resistant MRSA and had alternately Pseudomonas aeruginosa or linezolid-susceptible EMRSA-16 variant A1 isolated over 35 further months. CONCLUSIONS: Linezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance.

Evolución de los serotipos, fagotipos y resistencia a antimicrobianos de Salmonella sp en el departamento de salud 02 de la provincia de Castellón, España (2000-2006) / Evolution of serotypes, phagotypes and antibiotic resistance of Salmonella spp in the 02 health district of Castellón, Spain (2000-2006)

Retrospective study of serotypes, phage types and antibiotic resistance of Salmonella spp isolates in the 02 Health District of Castellón, Spain (2000-2006). Strains were serotyped using commercial sera, and they were tested for antimicrobial susceptibility with automated systems. Serotyping confirmation and phage typing were performed by the National Reference Laboratory. A total of 1505 strains were isolated, with 49 different serotypes, being the most frequent Enteritidis. The most common serotype/phage type combination was S. Enteritidis phagetype 1. Of the isolates 81.6 percent were susceptible to amoxicillin/clavulanic acid; 65.2 percent to ampicilin; 99.9 percent to ciprofloxa-cin; 93.4 percent to trimethoprim-sulphametoxazole; and 99.8 percent to cefotaxime. Molecular methods could be useful to complete epidemiologic studies since 25 percent of our isolates showed the same serotype/phage type combination. In our health district antimicrobial resistance in Salmonella is not an important problem.

Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases / Micobacteriófagos como herramientas versátiles para la manipulación genética y el desarrollo de métodos simples para el diagnóstico de enfermedades micobacterianas

Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over two million deaths per year worldwide. Due to its long doubling time (18 h), the microbiological detection of M. tuberculosis by conventional methods takes up to one month, unless the number of bacilli in the biological sample is high enough. Thus, drug resistance assessment requires at least one month for obtaining the primary culture and another month to determine its susceptibility to antimycobacterial drugs. Moreover, for a long time, the lack of genetic tools for mycobacteria has been a barrier for undertaking studies aimed at understanding the mechanisms of drug resistance and drug target identification, being all these topics of utmost importance considering the increase in the number of drug-resistant clones and the few therapeutic options available. Mycobacteriophages are promising as a novel source of genetic elements for mycobacteria manipulation, as well as for the development of versatile, simple, fast and cheap methods for drug resistance assessment of M. tuberculosis clinical isolates. We herein describe the background related to the use of mycobacteriophages, with emphasis placed on their utilization for drug resistance analysis in our country.

La tuberculosis, enfermedad causada por el bacilo Mycobacterium tuberculosis, es responsable de más de dos millones de muertes anuales en el mundo. Debido a su largo tiempo de duplicación (18 h), la detección bacteriológica de M. tuberculosis por métodos convencionales necesita de un mes o aun más, a menos que el número de bacilos en la muestra clínica sea suficientemente alto. Por consiguiente, se necesita un mínimo de dos meses para determinar la resistencia de este microorganismo a las drogas antituberculosas: uno para obtener el cultivo primario y otro para ensayar la sensibilidad frente a aquellas. La falta de herramientas para la manipulación genética de micobacterias ha dificultado la identificación de los blancos de acción de las drogas y el estudio de los mecanismos de resistencia a éstas, tópicos de la mayor relevancia dado el aumento mundial del número de aislamientos clínicos multirresistentes y las pocas opciones terapéuticas disponibles. Los micobacteriófagos son considerados nuevas herramientas para la manipulación de las micobacterias, así como para el desarrollo de métodos simples, rápidos y económicos para determinar la sensibilidad a drogas de los aislamientos clínicos de M. tuberculosis. En esta revisión se describen los antecedentes del uso de micobacteriófagos con énfasis en su utilización para el análisis de resistencia a drogas antituberculosas en nuestro país.

[Staphylococcus aureus prevalence among hospitalized patients]. / Staphylococcus aureus paplitimas hospitalizavimo laikotarpiu.

OBJECTIVE. To determine the prevalence of Staphylococcus aureus strains among hospitalized patients at the beginning of their hospitalization and during their treatment and the resistance of strains to antibiotics, and to evaluate epidemiologic characteristics of these strains. PATIENTS AND METHODS. Sixty-one patients treated at the Department of Cardiac, Thoracic and Vascular Surgery were examined. Identification of Staphylococcus aureus strains was performed using plasmacoagulase and DNase tests. The resistance of Staphylococcus aureus to antibiotics, beta-lactamase production, phagotypes, and phagogroups were determined. The isolated Staphylococcus aureus strains were tested for resistance to methicillin by performing disc diffusion method using commercial discs (Oxoid) (methicillin 5 microg per disk and oxacillin 1 microg per disk). RESULTS. A total of 297 Staphylococcus aureus strains were isolated. On the first day of hospitalization, the prevalence rate of Staphylococcus aureus strains among patients was 67.3%, and it statistically significantly increased to 91.8% on days 7-10 of hospitalization (P<0.05). During hospitalization, patients were colonized with Staphylococcus aureus strains resistant to cephalothin (17.6% of patients, P<0.05), cefazolin (14.6%, P<0.05), tetracycline (15.0%, P<0.05), gentamicin (37.7%, P<0.001), doxycycline (30.7%, P<0.001), and tobramycin (10.6%, P>0.05). Three patients (4.9%) were colonized with methicillin-resistant Staphylococcus aureus strains, belonging to phage group II phage type 3A and phage group III phage types 83A and 77; 22.6-25.5% of Staphylococcus aureus strains were nontypable. During hospitalization, the prevalence rate of phage group II Staphylococcus aureus strains decreased from 39.6% to 5.7% (P<0.05) and the prevalence rate of phage group III Staphylococcus aureus strains increased to 29.5% (P<0.001). CONCLUSIONS. Although our understanding of Staphylococcus aureus is increasing, well-designed community-based studies with adequate risk factor analysis are required to elucidate further the epidemiology of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Surveillance of methicillin-resistant Staphylococcus aureus provides relevant information on the extent of the methicillin-resistant Staphylococcus aureus epidemic, identifies priorities for infection control and the need for adjustments in antimicrobial drug policy, and guides intervention programs.

Characterization of Vibrio cholerae O1 ElTor typing phage S5.

S5 (ATCC No. 51352-B2), a Vibrio cholerae O1 ElTor typing phage was characterized. The growth characteristics and inactivation kinetics (thermal, UV and pH) of this lytic phage were investigated. Phage morphology was examined by electron microscopy and was classified as belonging to the family Podoviridae. The S5 phage genome is shown to be a linear double-stranded 39-kb-long DNA as determined by electron microscopy and restriction digestion. Partial denaturation maps were constructed and were used to show that the DNA is non-permuted and terminally redundant. The replication origin of this T7-like phage was visualized by electron microscopy. The polarity of packaging of S5 DNA in the phage head was determined. SDS-PAGE of phage S5 shows two major structural polypeptides of 50 and 42 kDa. A 3D structure of the phage head was reconstructed at a resolution of 37 A using Cryo-EM and a single-particle reconstruction technique.

Caracterización de dos brotes de fiebre tifoidea en Apartadó, Antioquia, 2005 / Characterization of two typhoid fever outbreaks in Apartadó, Antioquia, 2005

Introducción. La caracterización de los brotes de fiebre tifoidea es importante epidemiológicamente, debido a que esto permite la búsqueda de la fuente y el desarrollo de medidas de control. Objetivo. Describir un brote de fiebre tifoidea en el municipio de Apartadó y caracterizar fenotípica y genotípicamente los aislamientos de Salmonella Typhi relacionados con él. Materiales y métodos. Se estudiaron 44 pacientes, a 15 de ellos se les tomaron muestras para hemocultivos y a 7, muestras para coprocultivos. Los aislamientos bacterianos se estudiaron con pruebas bioquímicas y serotipificación y se determinó el perfil de susceptibilidad a antibióticos. Los aislamientos se evaluaron fenotípicamente por reacción en cadena de la polimerasa para los genes hilA, invA e IS- 200, y por electroforesis en campo pulsado con XbaI. Se estudiaron ocho muestras de agua asociadas al brote por reacción en cadena de la polimerasa y cultivo para la búsqueda de Salmonella. Resultados. A 15/44 pacientes se les confirmó el diagnóstico clínico de fiebre tifoidea, a 13 por hemocultivos y a 2 por coprocultivos positivos para S. Typhi. Todos los aislamientos de S. Typhi fueron sensibles a los antibióticos probados. La reacción en cadena de la polimerasa confirmó la presencia de los genes hilA y invA e IS-200 en todos los aislamientos estudiados. La electroforesis en campo pulsado agrupó 10 aislamientos en el patrón COINJPP.X01.0035, tres en el patrón COINJPPX01.0002, uno COINJPP.X01.0012 y uno COINJPPX01.0037. El estudio de aguas fue negativo para Salmonella spp. Conclusiones. La electroforesis en campo pulsado estableció la presencia de dos brotes, que inicialmente, por epidemiología y pruebas fenotípicas del patógeno, habían sido descritos como uno solo. Además, permitió diferenciar dos aislamientos de origen clonal diferente, que indicaron casos aislados. No se pudo corroborar la fuente de infección en el agua.

Introduction. The characterization of typhoid fever outbreaks is important because it is necessary to find the source of the infection and development control measures. Objective. A typhoid fever outbreak is described from Apartadó and the Salmonella Typhi isolates characterized by phenotypic and genotypic methods. Materials and methods. From 44 patients, 15 blood cultures and 7 stools cultures were recovered. Phenotypic identification of isolates was done by biochemical and serological tests, and antibiotic susceptibility was tested. Genes hilA, invA and the IS200 marker were evaluated by polymerase chain reaction; pulsed field gel electrophoresis was used for the XbaI gene. Eight water samples were examined by polymerase chain reaction and culture methods in order to isolate Salmonella spp. Results. Fifteen patients were confirmed for typhoid fever, 13 by blood cultures and two by stools cultures. All S. Typhi isolates were susceptible to the antimicrobials tested. The presence of hilA, invA and IS200 were confirmed by polymerase chain reaction in all isolates. The pulsed field gel electrophoresis method grouped 10 isolates in COINJPP.X01.0035 pattern, three in COINJPPX01.0002, one in COINJPP.X01.0012 and one in COINJPPX01.0037. Water isolates were negatives for Salmonella spp. Conclusions. Pulsed field gel electrophoresis discriminated the isolates in two outbreaks. Initially the cases were described as only one outbreak, by epidemiological criteria and phenotypic test. Additionally two isolates with different clonal origin were discriminated, indicating that they were unrelated to the other cases. It was not possible to confirm the infection source from water samples.

The presence of major world-wide clones for phage type 4 and 8 Salmonella enterica serovar Enteritidis and the evaluation of their virulence levels by invasiveness assays in vitro and in vivo.

Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.

Antimicrobial susceptibility, phage types, and pulsetypes of Salmonella Typhimurium, in São Paulo, Brazil

A total of 283 Salmonella Typhimurium strains isolated from cases of human infections and non human sources, were examined for antimicrobial susceptibilityand the incidence of resistance was 38 percent and multiple resistance (to three or more antimicrobials) was 15 percent. All 43 multidrug-resistant strains (MDR) and 13 susceptible ones were characterized by phage typing and pulsed- field gel electrophoresis (PFGE). The strains encompassed 14 definitive phage types (DT), three were untypable (UT), and 18 atypicals or reaction does not conform (RDNC), which belonged to 21 PFGE patterns, A1-A21. The predominant phage types were DT49, DT193, and RDNC and two strains belonging to DT 104 and 104b were also identified. The most commum PFGE patterns were A1 and A8. Analysis by PFGE and phage typing demonstrated that the most of the MDR were multiclonal and association among multiresistance, phage typing, and PFGE patterns was not so significant.

Actinophages as indicators of actinomycete taxa in marine environments.

It is necessary to continue to screen for new metabolites and evaluate the potential of less known and new bacterial taxa so that new and improved compounds for future use against drug-resistant bacteria or for chemical modification may be developed. There has been considerable interest in the detection and identification of marine microorganisms since they have been reported to produce bioactive compounds ranging from antitumour to antibacterial and antiviral agents. In this study, an improved technique that involves the exploitation of marine actinophages as indicators of the marine actinomycete taxa and uses marine bacteriophages as tools to reduce the numbers of common marine bacteria, which impedes the growth of rare actinomycetes on isolation plates, has been applied. This technique reduced the numbers of colony forming units of unwanted bacteria on isolation plates and hence increased the chances of detecting novel marine actinomycete genera for isolation and subsequent screening for antiviral activity.

Homology between two different Salmonella phages: Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15 + phageepsilon34.

A distinguishing feature of many microorganisms, belonging to the Gram negative group of bacteria, is the presence of the lipopolysaccharide on their cell surface. Salmonella is a prominent member of this group of bacteria. Many Salmonella phages use the LPS as the initial receptor in the infection process and they can distinguish subtle changes in the LPS molecules. The phage protein that is responsible for recognition of these cells is the tail or tailspike protein (TSP). Those TSPs, which use LPS as a receptor, are prokaryotic LPS-binding proteins. As an initial step in using phage TSPs as model systems for a detailed molecular genetic analysis of protein-LPS interactions, a comparison of two phages and their TSPs from two different Salmonella bacterial viruses (phages), Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15 + phage epsilon34, is being carried out. This present study shows significant viral protein homology between many viral structural proteins from these two phages including their TSPs. Significantly this report suggests a general structural motif for part of the TSP of phages and suggests that a more detailed comparative analysis of these TSPs is warranted.

Fenotipos bioquímicos y fagotipos de cepas de Salmonella enteritidis aisladas en Antofagasta, 1997-2000 / Biochemical phenotypes and phage types of Salmonella enteriditis strains isolated in Antofagasta during the period 1997-2000

BACKGROUND: PhP-S48 (Phene Plate Techniques AB), a method based on biochemical phenotypes has been developed and used successfully to typify S enteritidis strains in epidemiological studies. AIM: To identify phenotypes of S enteritidis isolated from eggs, chicken meat and infected humans in Antofagasta during the period 1997-2000. MATERIAL AND METHODS: PhP-S48 and phage typing were used to identify phenotypes of 33 S enteritidis strains, sixteen isolated from poultry and 17 from clinical sources. S enteritidis ATCC17036 was used as control strain. RESULTS: Twelve biochemical phenotypes (BTs) including 4 common (C) and 8 single (S) were identified. BTs C1 y C3 containing 16 and 5 strains, respectively, accounted for 63.6 per cent of the isolates. BT C1 was found in poultry and human sources in the period 1997-2000, and BT C3 was isolated from humans, in the period 1999-2000. Using phage typing, 5 phage types (PT) and 3 strains could be not typed (NTs). PT1 and PT21 were the dominant phage types, with 14 and 13 strains respectively. Strains of PT1 were isolated from poultry and human sources in the period 1997-2000. PT21 was found in poultry samples in the period 1997-1998 and in clinical samples, in the period 1997-1998. Combination of biochemical phenotypes and phage typing divided the strains into 5 phenotypes (BT:PT). Two phenotypes were the most frequently isolated, phenotype C1:1 with 8 isolates found in eggs and humans in 1999, and phenotype C1:21 with 5 strains isolated in 1997-1999. CONCLUSIONS: These results indicate the presence of one persistent and one recently emerged phenotype among S enteritidis in Antofagasta, Chile. PhP-S48 also provided information about a relationship among the strains.